The present invention relates generally to ex vivo gene therapy and, more specifically, to an improved method for transducing cells with viral-vectors.
The introduction of therapeutic genes into patient cells is a promising approach for the treatment of human diseases such as inherited genetic disorders, cancer, infectious diseases and immune disorders. One approach for introducing therapeutic genes involves isolating a target cell population from an individual, transfering therapeutic genes into the cells while the cells are maintained in culture, testing and selecting for transduced cells, and then reintroducing the genetically engineered cells into a subject. This procedure, known as ex vivo gene therapy, is limited by the current inability to achieve high level gene transfer and expression in clinically relevant numbers of cultured cells.
Transfer of genes into cells can be accomplished by a number of physical and biological methods. Pure DNA will enter cells following electroporation or direct microinjection, or when the DNA is complexed with cationic lipids or calcium phosphate. However, these methods are generally too inefficient and labor intensive for clinical use.
A more efficient method of gene transfer for clinical applications involves transduction of cells by viral-vectors that are genetically engineered to serve as carriers of heterologous genes. The choice of viral-vector depends on the target cell type and transduction approach desired. For example, modified adenovirus and adeno-associated virus can be produced at very high titers and provide for transiently high levels of gene expression in target cells. Replication-defective retroviruses are also used clinically, primarily for transducing cells where stable integration into the host chromosomal DNA is desired. The transferred gene is faithfully replicated and expressed in the progeny of the retrovirally-transduced cells. Other viruses with tropisms for specific cell types and engineered hybrid viruses are also are used in ex vivo gene therapy applications.
Gene therapy is most beneficial when all or the majority of cells that are introduced into a subject contain the desired genetic modification. However, current strategies for transducing cells with viral-vectors have not achieved this goal. One means of improving transduction efficiency is to bring the cultured target cells and the viral-vector into close proximity. For example, co-cultivation of target cells with virus-producing cells has been used to achieve high-efficiency gene transfer. However, co-cultivation raises concerns about the safety of exposing cells that will be introduced into subjects to other cells, as well as concerns about the reproducibility of infection in such a co-culture system.
Ex vivo culture and transduction of cells to date has generally involved the use of culture plates and flasks composed of conventional polystyrene, which is a hard, gas-impermeable plastic. Conventional polystyrene culture vessels are by necessity tied to an open system for regulation of gases dissolved in the culture medium and for regulation of pH of the culture medium. Typically, these conventional culture vessels are maintained in an incubator filled with regulated concentrations of O.sub.2 and CO.sub.2 ; the caps or tops of the culture vessels must be offset from the main vessel, and thus open to the environment, in order to admit the ambient gas mixture. As a result, the cell culture is exposed to contamination from the environment.
Thus, there exists a need for improved methods for transducing cells for ex vivo gene therapy applications. The present invention satisfies this need and provides related advantages as well.